Hindawi Publishing Corporation
Journal of Drug Delivery
Volume 2014, Article ID 424697, 5 pages
http://dx.doi.org/10.1155/2014/424697
Research Article
Polypeptide Multilayer Self-Assembly Studied by Ellipsometry
Marina Craig,1,2 Krister Holmberg,1 Eric Le Ru,3 and Pablo Etchegoin3
1
Department of Chemical and Biological Engineering, Chalmers University of Technology, Sven Hultins Gata 12,
412 96 Gothenburg, Sweden
2
Mölnlycke Health Care, P.O. Box 130 80, 402 52 Gothenburg, Sweden
3
School of Physical and Chemical Sciences, Victoria University of Wellington, P.O. Box 600, Wellington 6140, New Zealand
Correspondence should be addressed to Marina Craig; [email protected]
Received 8 May 2013; Revised 21 December 2013; Accepted 31 December 2013; Published 10 February 2014
Academic Editor: J. Varshosaz
Copyright © 2014 Marina Craig et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
A polypeptide nanofilm made by layer-by-layer (LbL) self-assembly was built on a surface that mimics nonwoven, a material
commonly used in wound dressings. Poly-L-lysine (PLL) and poly-L-glutamic acid (PLGA) are the building blocks of the nanofilm,
which is intended as an enzymatically degradable lid for release of bactericides to chronic wounds. Chronic wounds often carry
infection originating from bacteria such as Staphylococcus aureus and a release system triggered by the degree of infection is of
interest. The dry nanofilm was studied with ellipsometry. The thickness of the nanofilm was 60% less in its dry state than in its wet
state. The measurements showed that a primer was not necessary to build a stable nanofilm, which is practically important in our
case because a nondegradable primer is highly unwanted in a wound care dressing. Added V8 (glutamyl endopeptidase) enzymes
only showed adsorption on the nanofilm at room temperature, indicating that the PLL/PLGA “lid” may remain intact until the
dressing has been filled with wound exudate at the elevated temperature typical of that of the wound.
1. Introduction
Self-assembly techniques have become increasingly popular
as a tool to create controlled release systems for drug delivery.
One such technique is layer-by-layer (LbL) self-assembly,
which was pioneered by Decher [1, 2]. Based on this method
targeted release of bactericides into an infected chronic
wound can be developed. Overexposure of drugs often causes
unwanted side effects, which may be avoided by regulating
the drug release. Within chronic wound care, the primary
function of a dressing is to absorb and retain wound exudate.
However, a secondary function in the form of drug release
may also play a crucial role for chronic wound healing. Many
chronic wounds carry a bacterial infection [3, 4] generating
high amounts of bacterial proteases [5] in the wound, which
often disturbs the natural healing cascade. Such an infection
is normally treated with oral antibiotics or with gels, creams,
or wound dressings containing antimicrobial substances [6].
These are often applied in excess and may generate resistant
bacteria [7, 8]. Unnecessary use of bactericides may be
avoided by using an “intelligent dressing,” consisting of an
absorbing nonwoven dressing material with a biodegradable
polypeptide lid [9]. Antimicrobial agents are incorporated
into the product in such a way that they are released into
the infected wound when the wound exudate penetrates
the material and the lid is degraded. The lid is made up
of a thin polypeptide film and the degradation is caused
by bacterial proteases from the wound, for example, from
Staphylococcus aureus. The release of active substances will
hence be triggered by the infection. The thin film is assembled
at pH 7.4 and consists of two oppositely charged polypeptides deposited as alternating layers. The polypeptides of
choice, cationic poly-L-lysine (PLL) and anionic poly-Lglutamic acid (PLGA), are both biocompatible and highly
biodegradable [10, 11]. The procedure is an example of the
LbL technique. Quartz crystal microbalance with dissipation
monitoring (QCM-D) has been used to study both the
build-up and the degradation of the polypeptide film [9].
The underlying substrate was a model surface tailored by
the use of self-assembling alkane thiols to give the same
wetting characteristics as an existing nonwoven material
2
(regenerated cellulose) used in wound dressings. However,
the QCM-D method gives quantitative information about
the wet film and the values obtained for adsorption and
desorption at the underlying surface reflect the total amount
of material on the surface, that is, the two polypeptides as
well as water incorporated into the polypeptide film. Since
we are interested in obtaining quantitative information about
the polypeptide layers per se, an alternative measurement
technique is needed and ellipsometry, which is an optical
method that records adsorption and desorption of organic
molecules at a surface and disregards the water that comes
along with the adsorbate, was chosen as a suitable method
for the purpose.
2. Experimental and Methods
2.1. Preparation of Tailored Gold Surfaces. Clean (acid etching) and ready to use 41 nm gold sensors (XanTec bioanalytics, Düsseldorf, Germany) were tailored to imitate a regenerated cellulose or nonwoven surface using a 2.0 mM mixture of 60 mol% 11-hydroxy-1-undecanethiol and 40 mol% 1undecanethiol (Sigma Aldrich) in 99.5% ethanol. The sensors
were immersed in the alkane thiol solution for 16–20 hours
before rinsing in 99.5% ethanol and drying with nitrogen
gas before use. Gold substrates used for a second or third
time were cleaned in a Plasma Etch situated in a cleanroom.
Chosen effect, gas, time/cycle, and amount of cycles were
100 W, O2 , 1 min 10 sec/cycle, and two cycles. Contact angle
measurements of the substrates were used as a control of the
cleanliness.
2.2. Preparation of PLL and PLGA Solutions and Multilayer
Build-Up. Poly-L-lysine (PLL) (MW = 30–70 kDa) and polyL-glutamic acid (PLGA) (MW = 50–100 kDa) were purchased
from Sigma Aldrich. A 0.1 mM buffer solution was prepared
from Tris-HCl (Sigma Aldrich) and ultrapure water (MilliQ resistivity > 18 MΩ cm). NaCl (Fluka) (0.1 M) was added
to the 0.1 mM Tris-HCl solution followed by addition of
either PLL or PLGA in an amount corresponding to 1 mg/mL.
Both polyelectrolyte solutions were placed on a magnetic
stirrer for 1 hr before use. All end solutions had a pH of
7.4. The SAM covered gold surfaces were used for layerby-layer (LbL) assembly of PLL/PLGA multilayers. Three
PLL/PLGA bilayers were deposited by immersion into each
polypeptide solution, respectively, with rinsing steps using
Tris-HCl buffer after each immersion. The first PLL layer
was immersed in solution for 30 mins allowing a stable
cationic foundation for the rest of the build-up, whereafter the
deposition and rinsing were performed with 10 min intervals.
All (PLL/PLGA)3 samples were dried in N2 gas and placed for
ellipsometry measurements in air and ambient temperature.
Also, all samples were stored in air and remeasured after 24
hours to make sure that the samples were as dry as possible.
2.3. LbL and PLL/PLGA Nanofilms. Self-assembly with the
LbL technique can be described by creating an overcompensated surface charge by depositing one charged polymer
on a surface which in turn enables further deposition of an
Journal of Drug Delivery
oppositely charged polymer and so on [12]. Films consisting
of three PLL/PLGA bilayers were formed in this study. Both
PLL and PLGA are weak polyelectrolytes and therefore create
dynamic layers, a process that needs strict control of several
parameters, for example, polyelectrolyte concentration, ionic
strength, and so forth [13, 14].
2.4. Preparation of Enzyme Solutions. Endoproteinase Glu-C
from Staphylococcus aureus V8 (type XVII-B) and the reference enzyme, trypsin from bovine pancreas (type I, ∼10,000
BAEE units/mg protein), were both purchased from Sigma
Aldrich. The preparations of enzyme solutions included 1 : 100
[9, 15] dilution with ultrapure water (MilliQ, resistivity >
18 MΩ cm) and were used within 15 mins to ensure highest
possible enzyme activity. Samples with three PLL/PLGA
bilayers were immersed into the enzyme solutions, respectively, for possible degradation of peptides.
2.5. Ellipsometry. Ellipsometry was performed at ambient
temperature on an instrument from Beaglehole Instruments
(New Zealand). More information about the measuring
procedure can be found in the Supporting Information
(see Supplementary Material available online at http://dx.doi
.org/10.1155/2014/424697).
3. Results and Discussion
3.1. Ellipsometry Study and Data Analysis of the Build-Up of
the PLL/PLGA Multilayer. Three PLL/PLGA bilayers were
assembled directly on the alkane thiol model surface without
the use of a primer. A primer such as poly(ethylene imine)
is often used for LbL assemblies of polypeptides [16] but for
this specific application it is vital that all components are
susceptible to peptidase-catalyzed degradation and also are
biocompatible. Thus, we chose not to involve a primer. The
clean gold surface was initially studied in the ellipsometer.
Using the TFCompanion software and a double-layer model
with the Marquardt-Levenberg algorithm, a thickness of
406 ± 6.7 Å was obtained, which agreed well with the manufacturer’s specification of a thickness of 410 Å. At 633 nm
the 𝑛 for gold was 0.182 and 𝑘 was 3.436. It is known that
cleaning the gold by plasma treatment can cause an increase
of the surface roughness. The gold substrates were therefore
always remeasured after cleaning and fitted to the two-layer
model using a Bruggeman effective medium approximation
(EMA) layer on top of the bulk layer. However, the surface
roughness was estimated to be very small even after plasma
treatment, with a thickness of 371 ± 6.9 Å. Care was taken
that the values were repeatable throughout each test session.
The next step was to perform ellipsometry measurements on
the SAM coated surface. It was not considered appropriate to
add an EMA layer on top of a thin monolayer, which is why a
two-layer model for SAM on gold was used with 𝑛 assumed to
be 1.4999 at 633 nm [17]. The error of using an 𝑛 value of 1.5 ±
0.05 is estimated to be less than 1 Å [18]. Again, the surface
roughness was very small. The SAM thickness was 18 ± 2 Å,
which was appropriate since the theoretical value is 17 Å [19].
This surface is then the starting point for the LbL assembly
3.2. Ellipsometry Study and Data Analysis of the Enzymatic
Degradation of the PLL/PLGA Multilayers. After the ellipsometry measurement the (Au-SAM)-(PLL/PLGA)3 substrate was immersed in buffer solution and allowed to
swell for at least 30 minutes before use. The samples were
then moved to a new container and a solution of either
trypsin (bovine) or V8 glutamyl endopeptidase (Staphylococcus aureus) was added. Two different degradation times
were chosen for each enzyme solution, 3 hours and 16 hours.
The enzymes were either added at one time or up to three
times during the degradation period. The samples were then
rinsed with Milli-Q water and dried in nitrogen gas before
ellipsometry measurements were performed. The assumed
𝑛 value was 1.48 at 633 nm for both enzymes [21] (𝑘 =
0), which is an appropriate value for proteins [22, 23]. As
can be seen from Figure 2, trypsin adsorbed readily to the
polypeptide surface and increased the thickness up to 5 times.
This indicates that the overall positively charged trypsin (pH
0.9
0.9
0.8
0.8
0.7
0.7
0.6
0.6
0.5
0.5
0.4
0.4
400
600
X
of the two polypeptides, the cationic PLL and the anionic
PLGA. These were assembled in three bilayers, always starting
with deposition of PLL on the SAM surface. Thus, the entire
composition on which the ellipsometry measurements were
conducted can be written (Au-SAM)-(PLL/PLGA)3 , where
the deposited polypeptide film thickness was solved by using
a four-layer model using polypeptide bulk and polypeptide
EMA layers on top. Considering the low amount of bilayers
assembled, the refractive index was assumed to be rather
low (𝑛 = 1.4) and 𝑘 = 0 at 633 nm for both polypeptides
[20]. In literature it has been described that the refractive
index increases with increasing bilayers, as is the case for the
PLL/PLGA film.
The ellipsometry raw data was exported to the TFCompanion software and models were created using raw data
originating from the gold substrates modified by alkane
thiols as the starting point. Both polypeptides are weak
polyelectrolytes and LbL films from such polymers have been
reported not to be rigid [20]. This was also seen when fitting
the raw data to the model. Air was added in an amount of
about 10% of the volume of the polypeptides as one film
component, which resulted in a MSE (mean squared error) of
0.011 for all measurements. The final fit of the model of choice
for the Au-SAM-(PLL/PLGA)3 film can be seen in Figure 1.
Three PLL/PLGA bilayers deposited on the alkane thiolcovered gold surface gave a dry thickness of approximately
40 ± 3.4 Å. A QCM-D study gave a thickness of the wet
(PLL/PLGA)3 film of 100 ± 10 Å [9]. Hence one may conclude
that about 60% of the wet layer consisted of water. This is
reasonable and agrees with previously reported values in the
literature. It has been claimed that the large amount of bound
water in the film is due to the polypeptides adsorbing in loops
and tails, which favors storage of water in the film [20]. It is
interesting that roughly the same dry content of the film is
obtained in this work, in which no primer was used in the
LbL process, as in earlier work with a primer present. Thus,
the primer seems not to be needed on the alkane thiol surface
used in the present systems, which, as discussed above, is a
considerable advantage from an application point of view.
3
Y
Journal of Drug Delivery
800
Wavelength (nm)
Y (meas)
Y (calc)
X (meas)
X (calc)
Figure 1: Measured data for 𝑋 (related to the real part) and
𝑌 (related to the imaginary part) fitted well with the calculated
data using TFCompanion. The filmstack consists of (Au-SAM)(PLL/PLGA)3 , including an air content of 10% in the polypeptide
layers. The polypeptide film has 𝑛 = 1.4 at 633 nm. The calculation is
performed using the Marquardt-Levenberg and a three-layer model.
Ellipsometry equations for 𝑋 and 𝑌 can be found in Supporting
Information.
7.4) adsorbs readily on top of the thin polypeptide film
(negatively charged surface) but the adsorption evidently
does not result in any visible enzymatic degradation. This
is similar to what was seen in a QCM-D study of the same
system [9]. Despite a large increase in thickness it was difficult
to rule out any catalytic activity; however, as trypsin prefers
to catalyze positively charged substrates, the polypeptide film
ending with negatively charged PLGA would not be ideal
for catalysis. When modeling the adsorbed trypsin, a fivelayer model was used; however, only the thicknesses of the
trypsin and trypsin EMA layers were variable. The roughness
of the film was very low ending up with a trypsin thickness of
65 ± 3.7 Å (MSE = 0.013), that is, a total polypeptide/enzyme
thickness of 105 ± 3.7 Å.
The bacterial protease V8 behaved differently. The thickness of the nanofilm did not change much on exposure to the
solution of V8 enzyme, as can also be seen from Figure 2. A
four-layer model was used for the V8 enzyme’s adsorption
to the film, since the layer on top of the polypeptide film
was very thin. The best fit received a polypeptide/enzyme
thickness of 42 ± 2.9 Å. The immediate interpretation of
this result is that the V8 enzyme did not interact with
the polypeptide film, as both enzyme and the polypeptide
surfaces were negatively charged. However, Craig et al.
showed that this enzyme catalyzed degradation of the LbL
film provided that it was terminated by the anionic PLGA.
The reason for this is that the V8 peptidase is known to
be reactive in catalyzing cleavage of Glu-X bonds, that is,
peptide bonds involving a glutamate residue [24, 25]. The LbL
film studied in this work had PLGA as the terminating layer.
However, the temperatures differed. Whereas the QCM-D
measurements were performed at 32∘ C, which was intended
4
Journal of Drug Delivery
Trypsin
20
However, the present ellipsometry study was distinguished by
being conducted at ambient temperature, whilst a previous
study was performed at 32∘ C [9], a temperature chosen
to mimic the temperature of the wound. This difference
with respect to temperature dependency indicates that the
PLL/PLGA “lid” may remain intact until the dressing has
been filled with wound exudate with the elevated temperature
typical of that of the wound.
18
Thickness (nm)
16
14
12
10
8
6
4
V8
Nanofilm
2
0
1
2
3
Figure 2: The dry nanofilm thickness of 40 ± 3.4 Å nm increased
considerably when bovine trypsin adsorbed to the film surface,
whereas it remained less affected when exposed to the V8 enzyme,
at ambient temperature.
to mimic the temperature of a typical wound, the ellipsometry
experiments were performed at ambient temperature. Thus,
the temperature is vital and no or little enzymatic degradation occurred at room temperature. This is a practically
important piece of information because it indicates that the
wound dressing with the antimicrobial agents covered by
the polypeptide lid remains intact until it is contacted by
the exudate from a chronic wound at the approximate skin
temperature of 32∘ C. However, the V8 protease may not be
entirely inactive also at ambient temperature. The surface that
has been exposed to the V8 peptidase solution seems to be
slightly rougher immediately after the treatment (±6 Å) than
after one or two days, when all measuring points ended up at
the same value (±1 Å). This induced roughness of the surface
may indicate enzymatic cleavage of the top layer of the film,
that is, predominantly of PLGA.
4. Conclusion
The (PLL/PLGA)3 nanofilm was measured with ellipsometry
to study the thickness in its dry state. When comparing
with the film’s wet and dry thicknesses, it is clear that
about 60% of the wet film consists of water. This result is
in accordance with previously reported values from similar
systems despite the fact that in the present investigation
the polypeptides were adsorbed directly to a tailored gold
surface imitating nonwoven and not to a surface treated with
a primer such as PEI, which is the normal procedure. This
indicates that the character of the film without primer is
similar to that with primer. This is practically important in
our case because a nondegradable primer is highly unwanted
in a wound care dressing. The enzymatic degradation of
the nanofilm was also monitored by ellipsometry. Bovine
trypsin adsorbed at the polypeptide surface but there were
no indications of an enzymatic degradation of the LbL film
even after sequential addition of the peptidase. Also the V8
glutamyl endopeptidase from Staphylococcus aureus seemed
not to cause much degradation of the polypeptide nanofilm.
Conflict of Interests
The authors declare that there is no conflict of interests
regarding the publication of this paper.
Acknowledgments
This work has been performed within the VINN Excellence
Center SuMo Biomaterials, a center with financial support
from the Swedish governmental funding agency Vinnova
and from eight companies: AkzoNobel, AstraZeneca, Bohus
Biotech, Lantmännen, Mölnlycke Health Care, SCA Hygiene
Products, Södra Cell, and Tetrapak. The authors are grateful
to Mölnlycke Health Care and the research school BIOSUM
for economic support. They would also want to thank Dr.
Stefan Meyer for helping with software and Dr. Natalie Plank
for valuable help with the plasma treatment. This article is
dedicated to the memory of Professor Pablo Etchegoin.
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